Project Summary Gradual loss of brain function and neurodegeneration are common features of aging throughout diverse phyla. Understanding how the central nervous system (CNS) regenerates neurons throughout its life is a major area of interest in regenerative medicine. The study of neural stem cells (NSC) and CNS development and biology has been an active field of research, however, our knowledge of the precise developmental programs that regulate NSC dynamics during aging is limited due to the rarity of long term NSCs, the difficulty of monitoring NSCs in- vivo, and the incredible complexity of mouse and human brains. In this proposal we seek to understand basic principles and evolutionary conserved elements of neuronal regeneration, degeneration and aging using Botryllus schlosseri, a primitive chordate with a simple CNS that exhibits assayable and frequent (weekly) CNS tissue regeneration and degeneration throughout adult life, that can be monitored in vivo thanks to its nearly transparent body. These organisms can reproduce either sexually through gametes, or asexually through a stem cell mediated budding process. As a new generation of buds develop into mature individuals (zooids) the bodies of the old zooids undergo a synchronized wave of programmed cell death. During this weekly regeneration and degeneration cycle new brains form within the young buds in concert with the destruction of the old zooids? brain (Fig 1). This model system offers a unique opportunity to study the cellular and molecular mechanisms that direct weekly cycles of CNS generation and degeneration in young and old colonies (e.g. <3 months vs. >9 years) and to identify mutations that accumulate in the DNA of its CNS, stem cells that persist throughout its life. The Botryllus genome encodes hundreds of brain-associated genes with mammalian homologs. We have undertaken a systematic molecular (transcriptomic), cellular (FACS) morphological and behavioral characterization of old and young colonies (Fig 2). Botryllus transcriptome analyses revealed 393 genes that correlate with Alzheimer?s disease are differentially expressed between young and old colonies (Fig 2C-D). Blood analysis showed an increased frequency of phagocytic cells in old Botryllus colonies (Fig 2A-B), analogous to the age-associated shift in mouse and human HSC to favor myeloid cells. Morphological and functional analysis found that the brains of old colonies are smaller, contain a lower number of cells and have reduced response to stimuli (Fig 2E-F). Since stem cells are the only cells that self-renew and are maintained throughout the colony?s life, we hypothesize that genetic mutations that accumulate over time in NSC are the main cause of age associated neurodegenerative diseases. To test this hypothesis, we plan to characterize the molecular and cellular diversity of the Botryllus brain in young and old colonies, isolate its NSCs, identify mutations that accumulate in NSC and progenitor cells DNA, and test their effect on brain regeneration and function.